Core D will provide a number of chemical tools to aid the program project on proteases in cancer biology and as targets for therapy. (1) Core D will prepare positional scanning substrate libraries or substrate arrays to establish the substrate specificity profiles of cancer relevant proteases. This information facilitates the identification of physiological substrates of proteases and provides the basis for the design of selective substrates and potent inhibitors. (2) Core D will provide synthetic inhibitors of targeted proteases as required by the projects to study the cancer relevance of the targeted proteases. Two methods will be employed to access potent and selective inhibitors. First, if acceptable synthetic inhibitors to a protease target have already been developed by pharmaceutical companies, but are not available, then these inhibitors will be synthesized. Second, potent and selective inhibitors will be identified form mechanism-based inhibitor libraries based upon novel zinc binding groups. (3) Core D will develop methods to prepare fluorescent-tagged mechanism-based irreversible inhibitors of cancer relevant proteases for identifying and quantifying active proteases in tissue samples. The methods for fluorescent tagging could conceivably be extended to MRI applications.